Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
  • C12N2310/121—Hammerhead
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
  • C12N2310/128—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes processing or releasing ribozyme

Definitions

• the present invention relates to the field of molecular biology. Particularly, the present invention relates to the field of artificial ribozymes.
• RNA and DNA can catalyze a broad range of chemical reactions.
• Nucleic acids can fold into complex three-dimensional structures including binding sites and catalytic centers and provide an environment in which various reactions can be facilitated.
• Extensive research in this field has shown that RNA and DNA are able to accelerate the formation, cleavage and rearrangement of various types of covalent bonds.
• a catalytic ribozyme refers to an RNA having an enzyme activity (catalytic ability), i.e., an RNA catalyst.
• the term ribozyme is a fusion of two words, ribonucleic acid (RNA) and enzyme.
• RNA ribonucleic acid
• the so-called "hammerhead" structures are a class of naturally occurring RNAs structures which undergo site specific autolytic cleavage, as they consist of a small catalytic RNA motif capable of endonucleolytic (self-) cleavage.
• RNAs originates from satellite RNAs of plant viruses, and they are composed of a catalytic core of conserved nucleotides flanked by three helices, two of which form essential tertiary interactions for fast self-scission under physiological conditions. Its presence in several eukaryotic genomes has been widely reported since their discovery.
• RNA which can be cleaved is considered the substrate (target RNA) and the RNA which catalyzes the cleavage, is designated "ribozyme".
• Ribozymes assuming a hammerhead structure are designated "hammerhead ribozymes”.
• Ribozymes are being developed as treatments for a variety of diseases ranging from inborn metabolic disorders to viral infections and acquired diseases such as cancer. Ribozymes can be used both to down-regulate and to repair pathogenic genes. However, although some variations on naturally occurring ribozymes are available, they have not been very effective in mammalian cells.
• the present invention provides:
• the term “about” as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. For instance, the term “about” means the indicated value ⁇ 1% of its value, or the term “about” means the indicated value ⁇ 2% of its value, or the term “about” means the indicated value ⁇ 5% of its value, the term “about” means the indicated value ⁇ 10% of its value, or the term “about” means the indicated value ⁇ 20% of its value, or the term “about” means the indicated value ⁇ 30% of its value; preferably the term “about” means exactly the indicated value ( ⁇ 0%).
• the conjunctive term "and/or" between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by "and/or", a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term "and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term "and/or.”
• plural is referred herein as two or more nucleic acid molecules, including DNA, RNA, and DNA/RNA molecules.
• polynucleotide refers to a polymer of at least 8 nucleotides in length selected from ribonucleotides, deoxyribonucleotides, or modified forms thereof. If the polynucleotide comprises ribonucleotides, it is referred to as "polyribonucleotide”. When a polyribonucleotide has the ability of carrying out enzymatic catalysis or cleavage over a substrate polyribonucleotide, it is called herein a “catalytic polyribonucleotide".
• catalytic polyribonucleotide or refers to an RNA sequence (RNA enzyme) capable of cleaving a substrate polyribonucleotide when it hybridizes to it.
• RNA sequence RNA enzyme
• capable of cleaving or “capable of exerting a catalytic cleavage activity” refers to an enzymatic reaction carried out by a catalytic polyribonucleotide over a substrate polyribonucleotide, that results in the cleavage of said substrate polyribonucleotide.
• substrate polyribonucleotide refers to a polyribonucleotide molecule that is cleaved by the action of the catalytic polyribonucleotide.
• a "self-catalytic polyribonucleotide” or “self-cleaving polyribonucleotide” refers to a single polyribonucleotide molecule having a RNA sequence comprising at least two regions, wherein a first region comprises a catalytic polyribonucleotide as defined above, and the second region comprises a substrate polyribonucleotide, as also defined above, and that is cleaved by the catalytic polyribonucleotide.
• the first and second regions comprised in the molecule may be connected by a linker, preferably a linker polyribonucleotide sequence.
• self-catalytic or self-cleaving polyribonucleotide is also called herein "self-catalytic ribozyme” or “self-cleaving ribozyme”.
• self-catalytic and self-cleaving are considered synonymous herein.
• RNA molecules or polyribonucleotides are defined herein: those called herein “catalytic polyribonucleotide” that comprise the sequence of the catalytic polyribonucleotide but do not comprise the sequence of the substrate polyribonucleotide, and those molecules called herein "self-catalytic polyribonucleotide", “self-cleaving polyribonucleotide”, “self-cleaving ribozyme” or “self-catalytic ribozyme” that have both sequences, namely the catalytic polyribonucleotide and the substrate polyribonucleotide.
• catalytic polyribonucleotide that comprise the sequence of the catalytic polyribonucleotide but do not comprise the sequence of the substrate polyribonucleotide
• self-catalytic polyribonucleotide self-catalytic polyribonucleotide
• self-cleaving polyribonucleotide self-
• nucleotides or nucleotide analogues is not limited in particular, and is meant to include deoxynucleoside triphosphates (dNTPs) such as for example, but not limited to dATP, dCTP, dGTP, dTTp, dITP, dUTP or derivatives thereof.
• dNTPs deoxynucleoside triphosphates
• derivatives reference is made to dideoxynucleotides such as ddATP, ddCTP, ddGTP, ddTTp, ddITP, ddUTP, or oxidized derivatives like 8oxoA, 8oxoG, 50HC, 5OHU, or labelled derivatives like fluorescent labelled derivatives or even more complex labels like the labelled used for third generation sequencing.
• dideoxynucleotides such as ddATP, ddCTP, ddGTP, ddTTp, ddITP, ddUTP, or oxidized derivatives like 8oxoA, 8oxoG, 50HC, 5OHU, or labelled derivatives like fluorescent labelled derivatives or even more complex labels like the labelled used for third generation sequencing.
• Example 1 shows the in vitro cleavage efficiency of 7 constructs, each with a different synthetic self-cleaving ribozyme in the 5' region.
• a comparison is made between 4 previously known naturally occurring hammerhead ribozymes (from Ricordea florida, Trichobilharzia regent and Schistosoma rodhaini -2 sequences-) and three new artificial designed ribozymes.
• the three artificial self-cleaving ribozymes showed better self-cleavage than the natural occurring ones, resulting in a RNA fragment of approximately 300 nt.
• Example 2 shows the in vitro cleavage efficiency of 7 precursor circRNAs each flanked by one of the self-cleaving ribozymes analyzed in example 1. As shown in Figure 2 , the three precursor circRNA containing the three artificial self-cleaving ribozymes showed a double self-cleavage resulting in a circRNA fragment of approximately 300 nt.
• Example 3 shows the circularization efficiency of 7 precursor circRNAs from Example 2, each flanked by one of the 5' HH ribozymes analyzed in example 1 and the HH ELVd ribozyme in the 3' region.
• the three precursor circRNA containing the three artificial self-cleaving ribozymes showed a 100% circularization efficiency, which is a higher efficiency than the natural occurring ribozymes.
• the present invention provides polynucleotides that are capable of cleaving substrate polynucleotides.
• the polynucleotides provided herein are catalytic polyribonucleotides
• the substrate polynucleotides are substrate polyribonucleotides.
• the catalytic polyribonucleotides provided herein are synthetic.
• Synthetic catalytic polyribonucleotide means a catalytic polyribonucleotide which is not a naturally-occurring RNA catalyst, although they can be truncated or altered versions of naturally-occurring ribozymes.
• synthetic catalytic polyribonucleotide also includes catalysts synthesized in vitro and catalysts synthesized in vivo.
• synthetic is synonymous to “artificial”, i.e., they are human made catalytic polyribonucleotides or ribozymes.
• three artificial catalytic polyribonucleotides were designed in the present invention.
• the sequences of them correspond to SEQ ID NOs: 23, 25 and 27, wherein their substrate polyribonucleotide correspond to SEQ ID NO: 24, 26 and 28, respectively.
• the polyribonucleotide sequences of said catalytic polyribonucleotides can be grouped and represented by the general formula as defined in SEQ ID NO: 33, wherein the substrate polyribonucleotide corresponds to the general formula of SEQ ID NO: 20.
• a first aspect of the present invention provides a synthetic catalytic polyribonucleotide, also referred as the "catalytic polyribonucleotide of the invention", comprising or consisting of SEQ ID NO: 33, wherein N 1 , N 2 , can be any nucleotide provided that N 1 in SEQ ID NO: 33 is capable of base pair with N 4 in SEQ ID NO: 20, and provided that N 2 in SEQ ID NO: 33 is capable of base pair with N 3 in SEQ ID NO: 20.
• said synthetic catalytic polyribonucleotide of SEQ ID NO: 33 is capable of exerting a catalytic cleavage activity on a substrate polyribonucleotide having the sequence of SEQ ID NO: 20, wherein N 3 , N 4 , can be any nucleotide, provided that N 4 in SEQ ID NO: 20 is capable of base pair with N 1 in SEQ ID NO: 33, and provided that N 3 in SEQ ID NO: 20 is capable of base pair with N 2 in SEQ ID NO: 33.
• the nucleotides N 1 , N 2 , N 3 , and N 4 can be any nucleotides, provided that N 1 is capable of base pair with N 4 and N 2 is capable of base pair with N 3 .
• a "base pair” refers to a specific combination of two nucleotides of nucleic acids connected via hydrogen bonds. A combination of nucleotides capable of forming a base pair are said to be "complementary" to each other.
• A adenine (A) pairs with thymine (T) and guanine (G) pairs with cytosine (C)
• RNA A pairs with uracil(U) and G pairs with C.
• so-called non-Watson-Crick base pairs such as G-A, G-U also occur as thermodynamically stable base pairs, and these combinations are also said to be “complementary” herein.
• the synthetic catalytic polyribonucleotide of the invention comprises or consists of SEQ ID NO: 23.
• the synthetic catalytic polyribonucleotide of the invention comprises or consists of SEQ ID NO: 23, wherein said synthetic catalytic polyribonucleotide is capable of exerting a catalytic cleavage activity on a substrate polyribonucleotide having the base sequence of SEQ ID NO: 24.
• the synthetic catalytic polyribonucleotide of the invention comprises or consists of SEQ ID NO: 25.
• the synthetic catalytic polyribonucleotide of the invention comprises or consists of SEQ ID NO: 25, wherein said synthetic catalytic polyribonucleotide is capable of exerting a catalytic cleavage activity on a substrate polyribonucleotide having the base sequence of SEQ ID NO: 26.
• the synthetic catalytic polyribonucleotide of the invention comprises or consists of SEQ ID NO: 27.
• the synthetic catalytic polyribonucleotide of the invention comprises or consists of SEQ ID NO: 27, wherein said synthetic catalytic polyribonucleotide is capable of exerting a catalytic cleavage activity on a substrate polyribonucleotide having the base sequence of SEQ ID NO: 28.
• the catalytic polyribonucleotides according to the invention are synthetic or artificial catalytic RNA sequences that are capable of recognizing and cleaving a substrate polyribonucleotide.
• the catalytic cleavage may occur in cis or trans, depending on whether the substrate polyribonucleotide is part of the same molecule as the catalytic polyribonucleotide (cis cleavage) or is part of a different molecule (trans cleavage).
• a cleavage "in cis” means a cleavage happening locally within the same molecule (i.e., intramolecular cleavage).
• a cleavage "in trans” means that the cleavage is happening in a different molecule (i.e., intermolecular cleavage).
• the synthetic catalytic polyribonucleotides of the invention are capable of cleaving a substrate polyribonucleotide comprised in a nucleic acid molecule that is different from the molecule where said catalytic polyribonucleotide of the invention is comprised.
• the substrate polyribonucleotide is comprised in a nucleic acid of interest, such as the transcriptome of an organism.
• Said organism can be a virus, a bacteria, a fungi, or a eukaryotic cell.
• said transcriptome is the RNA or mRNA molecules expressed by said organism.
• the substrate polyribonucleotide in the case of a trans cleavage is a polyribonucleotide sequence, preferably a mRNA sequence, comprised in the transcriptome of a human pathogen.
• the synthetic catalytic polyribonucleotide and its corresponding substrate polyribonucleotide are comprised in the same molecule, thereby forming a molecule that has a self-cleavage ability, named herein a self-catalytic polyribonucleotide or self-catalytic ribozyme.
• Said self-catalytic polyribonucleotide/ribozyme is characterized by comprising a catalytic polyribonucleotide and its corresponding substrate polyribonucleotide.
• the present invention also provides in the first aspect self-catalytic polyribonucleotides comprising at least two regions, wherein a first region comprises the catalytic polyribonucleotide of the invention (defined above), and the second region comprises its corresponding substrate polyribonucleotide.
• This molecule is also referred herein as the "self-catalytic polyribonucleotide of the invention", or “self-catalytic ribozyme of the invention”.
• the self-catalytic polyribonucleotides of the invention are synthetic, i.e., non-natural molecules.
• polyribonucleotide of the invention is used from herein after to refer to both the catalytic polyribonucleotides of the invention and the self-catalytic polyribonucleotides/ribozymes of the invention.
• the catalytic polyribonucleotide and the substrate polyribonucleotide comprised in the synthetic self-catalytic polyribonucleotide of the invention are connected by means of a linker.
• the linker is a nucleotide sequence, preferably GAUA.
• SEQ ID NOs: 29-31 represent examples of the synthetic self-catalytic polyribonucleotides of the invention where the catalytic polyribonucleotide and its corresponding substrate polyribonucleotide are comprised in the same molecule, thereby exerting a cis cleavage.
• Said synthetic self-catalytic polyribonucleotides can be grouped and represented by the general sequence of SEQ ID NOs: 21 or 22.
• a synthetic self-catalytic polyribonucleotide that comprises or consists of SEQ ID NO: 21, wherein N 1 , N 2 , N 3 , and N 4 can be any nucleotides, provided that N 1 is capable of base pair with N 4 and N 2 is capable of base pair with N 3 , and wherein N 5 is a linker, preferably the nucleotide sequence "GAUA".
• the synthetic self-catalytic polyribonucleotide comprises or consists of SEQ ID NO: 22, wherein N 1 , N 2 , N 3 , and N 4 can be any nucleotides, provided that N 1 is capable of base pair with N 4 and N 2 is capable of base pair with N 3 .
• the synthetic self-catalytic polyribonucleotide comprises or consists of SEQ ID NO: 29, 30, or 31.
• These self-catalytic polyribonucleotides have a structure compatible with type I hammerhead-like ribozymes.
• a hammerhead ribozyme (HHR) is a small (50-150 nucleotides) catalytic RNA motif capable of endonucleolytic (self-) cleavage. It is composed of a catalytic core of conserved nucleotides flanked by three helices (helices I, II and III).
• HHRs that cleave other RNAs in trans have been identified, the majority of known natural HHRs are cis-, i.e., self-cleaving entities that contain the motif within a single RNA strand. This gives rise to the three circularly permuted forms that are named types I, II, or III, according to the open-ended helix that connects the motif with the flanking sequences.
• the synthetic catalytic polyribonucleotide of the invention is comprised in or is a portion of a larger RNA molecule.
• portion or region when referred to a nucleotide sequence means a nucleotide region or fragment of said sequence.
• the self-catalytic polyribonucleotides described above which are RNA molecules where a portion of said molecule comprises the catalytic polyribonucleotide of the invention and another portion comprises its substrate polyribonucleotide.
• An RNA molecule comprising at least one of the polyribonucleotides of the invention is referred herein as the "RNA molecule of the invention".
• the RNA molecule of the invention comprises at least one self-catalytic polyribonucleotide of the invention is called "self-catalytic RNA molecule of the invention” or "self-cleaving RNA molecule of the invention”.
• the RNA molecule of the invention comprises one or more of the polyribonucleotide of the invention in its 5' and/or 3' region. More preferably, the RNA molecule of the invention comprises a self-catalytic polyribonucleotide of the invention in its 5' and/or 3' end.
• the expression "3' region”, as used herein, refers to a region of a nucleotide strand that includes the 3' end of said strand. As used herein, the "3' region” and the "3' terminal region” refer to the same region of a nucleotide strand and are used interchangeably.
• the term "3' end”, as used herein, designates the end of a nucleotide strand that has the hydroxyl group of the third carbon in the sugar-ring of the ribose or deoxyribose at its terminus.
• the expression "5' region”, as used herein, refers to a region of a nucleotide strand that includes the 5' end of said strand. As used herein, the “5' region” and the “5' terminal region” refers to the same region of a nucleotide strand and may be used interchangeably.
• the term “5' end”, as used herein, designates the end of a nucleotide strand that has the fifth carbon in the sugar-ring of the deoxyribose at its terminus.
• the self-polyribonucleotide of the invention is placed in the 5' end of the RNA molecule of the invention where it is comprised.
• a nucleic acid molecule may comprise or encode for one or more of the polyribonucleotides, including the catalytic polyribonucleotides and/or the self-catalytic polyribonucleotides of the invention.
• a nucleic acid molecule may also comprise or encode for other self-catalytic ribozymes or other self-catalytic polyribonucleotides, so that they are used in combination.
• said other self-catalytic ribozymes or other self-catalytic polyribonucleotide that are used in combination with the polyribonucleotides of the invention are hammerhead ribozymes, preferably hammerhead type III ribozyme, most preferably Elvd.
• the RNA molecule of the invention comprises two self-catalytic polyribonucleotides of the invention, one in each end of the molecule.
• the RNA molecule of the invention comprises a first and a second self-catalytic polyribonucleotides, wherein the first self-catalytic polyribonucleotide is a self-catalytic polyribonucleotide of the invention, and the second self-catalytic polyribonucleotide is a known self-catalytic polyribonucleotide, such as a hammerhead ribozyme, preferably hammerhead type III ribozyme, most preferably Elvd.
• the term "known” as used herein refers to a self-catalytic ribozyme that either "natural", namely found in nature, or "synthetic", i.e., previously sequenced and characterized.
• RNA molecule of the invention comprises two self-catalytic polyribonucleotides
• it will be self-cleaved twice, resulting in three RNA molecules: a first RNA molecule corresponding to the 5' region of the original RNA molecule of the invention, a second RNA molecule corresponding to the region of the original RNA molecule that was flanked by the two self-catalytic polyribonucleotides, and a third RNA molecule corresponding to the 3' region of the original RNA molecule of the invention.
• the second RNA molecule may be subsequently circularized (i.e., by ligation using a suitable ligase), thereby forming a circular RNA (also called herein circRNA).
• the RNA molecule of the invention comprises, before cleavage, a first and a second self-catalytic polyribonucleotides, wherein:
• ELVd ribozyme is referred herein to the Eggplant latent viroid self-catalytic ribozyme or polyribonucleotide that consists of a single-stranded, circular, non-coding RNA of 332-335 nucleotides that folds in a branched quasi-rod-like minimum free-energy conformation.
• said ELVd self-catalytic polyribonucleotide/ribozyme comprises or consists of SEQ ID NO: 32.
• the present invention provides a self-cleaving RNA molecule of the invention that comprises, before cleavage, a first and a second self-catalytic polyribonucleotides or ribozymes, wherein:
• the self-cleaving RNA molecule of the invention that is the precursor of the circRNA of the invention does not necessarily need to have two self-catalytic polyribonucleotides. It is sufficient if said RNA molecule has only one self-catalytic polyribonucleotide, preferably a self-catalytic polyribonucleotide of the invention, provided that the RNA molecule comprises a compatible end capable of being ligated to the ends that said self-catalytic polyribonucleotide creates after its cleavage activity.
• the term "compatible ends capable of being ligated” refers to RNA overhangs that can be joined by ligation, i.e., that are ligatable ends.
• the present invention provides a self-cleaving RNA molecule of the invention that comprises, before cleavage, a first and a second regions, wherein:
• the present invention also provides a circular RNA molecule of the invention obtained or obtainable by the method comprising the steps of:
• a sequence capable of being ligated to the end generated after the self-cleavage of the self-catalytic polyribonucleotide of the first region refers to a sequence that has a compatible end with the end generated after the self-cleavage of the self-catalytic polyribonucleotide comprised in the first region.
• This sequence can be synthetised in vitro or may be generated by enzymatic reaction.
• RNA molecule to be cleaved
• suitable reaction conditions to promote that the self-catalytic polyribonucleotide comprised in the RNA molecule is automatically activated and carries out the self-cleavage. Said conditions are known in the art and are also exemplified in Examples 1-3 below.
• the circular RNA molecule of the invention obtained or obtainable by the method comprising the steps of:
• the circular RNA molecule of the invention obtained or obtainable by the method comprising the steps of:
• RtcB ligase is referred herein to as a ligase from E. coli that joins single stranded RNA with a 3'-phosphate or 2',3'-cyclic phosphate to another RNA with a 5' -hydroxyl.
• RNA molecules as defined in (i) to (vi) can be circularized using a suitable ligase, such as the RtcB ligase, thereby forming the circRNA of the invention.
• polyribonucleotides including the catalytic polyribonucleotides and the self-catalytic polyribonucleotides
• the polyribonucleotides of the invention can be synthesizing a RNA nucleic acid molecule having one or more polyribonucleotides explained above.
• the synthesis of the polyribonucleotide can be performed by any method commonly used by those skilled in the art.
• the self-catalytic polyribonucleotides of the invention can be produced in the form of a DNA molecule that encodes or that, upon transcription, produces, the polyribonucleotide of the invention, amplify it by PCR to prepare a template DNA and transcribe it by T7RNA polymerase to synthesize the intended polyribonucleotide.
• a DNA molecule that encodes the molecules (i) to (vi) may be the DNA molecules (also called circRNA precursors) of SEQ ID NOS: 16, 17 and 18.
• DNA molecule encoding for one or more of the polyribonucleotides defined above is also included in the present invention.
• This DNA molecule is also referred herein as the "DNA molecule of the invention”.
• Said DNA molecule of the invention produces, upon transcription, the polyribonucleotides of the invention.
• the DNA molecule of the invention comprises or consists of SEQ ID NOs: 5, 6, or 7.
• RNA molecule for instance the RNA molecule or the circRNA molecule of the invention, departing from a DNA molecule, a step of in vitro or in vivo transcription is needed. This is to generate the RNA molecule that will automatically be self-cleaved if the polyribonucleotides of the invention are comprised in said RNA molecule.
• the DNA molecule should thus be designed to include the necessary elements for said step of in vitro or in vivo transcription.
• the DNA molecule may comprise transcription initiation sites or a suitable promoter.
• the DNA molecule that is a precursor of the RNA of the invention comprises a suitable promoter such as T7 promoter.
• the DNA molecule encodes at least two self-cleaving ribozyme, wherein a first self-cleaving ribozyme is a self-catalytic polyribonucleotide of the invention, and a second self-cleaving ribozyme is a known (i.e., natural or synthetic) hammerhead ribozyme, preferably hammerhead type III ribozyme, most preferably Elvd, and wherein the sequence encoding the first self-catalytic polyribonucleotide of the invention is placed in the 5' region, preferably 5' end, of said DNA molecule, and the sequence encoding the second self-cleaving ribozyme is placed in the 3' region, preferably 3' end, of said DNA molecule.
• a first self-cleaving ribozyme is a self-catalytic polyribonucleotide of the invention
• a second self-cleaving ribozyme is a known (i.
• DNA molecule of the invention encodes or upon transcription results in the circRNA of the invention.
• This is exemplified in Examples 2 and 3, where seven DNA molecules were generated, of which three of them (SEQ ID NOs: 16, 17, and 18) are precursors molecule that, upon transcription, generate a RNA of the invention, that is subsequently cleaved and ligated, thereby resulting in the circular RNA of the invention. Therefore, provided herein are DNA molecules of the invention comprising or consisting of SEQ ID NO: 16, 17 or 18.
• the molecules (a) to (d) defined herein comprise or encode for any of SEQ ID NOs: 33, 21, 22, 23, 25, 27, 29, 30 or 31.
• the molecules (a) to (d) defined above may further comprise or encode for other ribozymes, such as hammerhead ribozymes, preferably hammerhead type III ribozyme, most preferably Elvd.
• compositions comprising one or more of the molecules defined herein, including the polyribonucleotides (catalytic polyribonucleotides and self-catalytic polyribonucleotides) of the invention, and DNA and RNA molecules encoding or comprising, respectively, thereof.
• kits comprising one or more of the molecules defined herein, including the polyribonucleotides (catalytic polyribonucleotides and self-catalytic polyribonucleotides) of the invention, and DNA and RNA molecules encoding or comprising, respectively, thereof.
• polyribonucleotides of the invention may be provided in the form of a system of two or more molecules that are capable of interacting with each other and give rise to the polyribonucleotides of the invention.
• a second aspect of the present invention refers to the use of the molecules and compositions as defined in the first aspect, in medicine or for the generation of a medicament.
• the polyribonucleotides (catalytic polyribonucleotides and self-catalytic polyribonucleotides) of the invention or compositions comprising thereof may be used in the therapeutic field as molecules capable of targeting sequences of interest, that are substrate polyribonucleotides.
• the synthetic catalytic polyribonucleotides are used in the medical field or in a method of treatment by therapy.
• a catalytic polyribonucleotide of the invention may be used to produce the trans cleavage of the transcriptome of a virus, if the transcriptome of said virus comprises its corresponding substrate polyribonucleotide, thereby inhibiting the viral replication. Therefore, the polyribonucleotides of the invention, particularly the catalytic polyribonucleotides of the invention, can be used in medicine.
• Catalytic ribozymes show great therapeutic promise for altering viral replication in vivo.
• a therapeutic ribozyme is exposed to the substrate polynucleotide in one of two general ways.
• the catalytic ribozyme may be isolated in the laboratory and packaged in a suitable delivery vehicle.
• the DNA encoding the ribozyme of interest is incorporated into a vector with a suitable promotor and delivered to the target cell.
• a third aspect of the present invention refers to an in vivo or in vitro method of cleaving a substrate polyribonucleotide by using one or more of the polyribonucleotides of the invention.
• the use refers to an in vitro or in vivo method for cleaving SEQ ID NO: 20, by using the catalytic polyribonucleotide of SEQ ID NO: 33, wherein N 1 , N 2 , N 3 , and N 4 can be any nucleotides, provided that N 1 is capable of base pair with N 4 and N 2 is capable to base pair with N 3 .
• the cleaved substrate polyribonucleotide obtained or obtainable from the method defined in said aspect.
• a fourth aspect of the present invention refers to a method to produce the circular RNA molecule of the invention, the method comprising the steps of:
• the self-catalytic polyribonucleotide that is comprised in the second region is a known (i.e., natural or synthetic) self-catalytic polyribonucleotide, such as a hammerhead ribozyme, preferably hammerhead type III ribozyme, most preferably Elvd.
• said ELVd comprises SEQ ID NO: 32.
• the first region of the RNA molecule generated in step a) is placed in the 5' region, preferably 5' end, of said molecule, and the second region is placed in the 3' region, preferably 3' end, of said molecule.
• the method to produce the circular RNA molecule of the invention comprises the steps of:
• the method to produce the circular RNA molecule of the invention comprises the comprising the steps of:
• Step a) in the method of the fourth aspect comprises or consists of providing a DNA molecule.
• This DNA molecule is designed so that, when it is transcribed in step b), a RNA molecule is formed.
• transcription initiation elements such as suitable promoters, preferably a T7 promoter.
• Step b) in the method of the fourth aspect comprises or consists of the in vitro or in vivo transcription of the DNA molecule provided in a), thereby producing an RNA molecule that is automatically self-cleaved by action of the self-catalytic polyribonucleotide of the first and second regions, if present, so that a RNA molecule with ligatable ends is obtained.
• a molecule with "ligatable ends” is a molecule whose 5' and 3' ends are compatible to be ligated by, e.g., action of a ligase. This step is common in the art and the skilled person knows how to provide the necessary reagents and conditions to promote the transcription of the DNA molecule provided in a).
• a transcription step is shown in Example 2 of the present invention, where the DNA molecule of step a) comprises a T7 promoter, and it is thus in vitro transcribed using T7 RNA polymerase. The resultant RNAs are then self-cleaved, as shown in Fig. 2 .
• step c) in the method of the fourth aspect comprises or consists of adding a ligase capable of joining the 5' and 3' end of the cleaved RNA molecule obtained in step b).
• the step of ligation the ends of an RNA molecule are known in the art.
• a ligation step is shown in Example 3 of the present invention, where the linear RNAs obtained from Example 2 are circularized by incubating them under suitable conditions (i.e., 37°c for 1 hour) with RtcB ligase.
• a further optional step d) may be purifying them or removing the non-ligated RNA molecules by incubating the sample with an RNAse, such as an RNAse R.
• RNA molecule or molecules obtained or obtainable from the method defined in said aspect.
• the invention provides a method to provide a plurality of RNA molecules characterized by having a sequence in the 5' region that is identical or substantially identical in all of the molecules.
• a region is "substantially identical" to another region when the percentage of nucleobase identity between both regions is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99,9%, preferably at least 99,99%.
• the "substantial identity” includes the possible errors (i.e., insertion, deletion or substitution of nucleotides made by polymerase enzymes or by DNA damage, library processing, sequencing or mapping.
• Identity means a percentage of identity of 100%.
• the "5' region” that is identical or substantially identical in the plurality of RNA molecule comprises about 60, 50, 40, 30, 25, 20 ,15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nucleotides from the 5' end of the RNA molecules.
• the sequence that is identical or substantially identical in the plurality of RNA molecules is located in the 5' end of each of said RNAs molecules.
• the plurality of RNA molecules obtained after the method of the fifth aspect all have the same sequence in their 5' region, preferably the same sequence in their 5' end. This is achieved following the method steps of:
• the self-catalytic polyribonucleotide of the invention comprised in the RNA molecules produced in step a) is placed in the 5' region, preferably 5' end, of the each of the RNA molecules.
• the method according to the fifth aspect comprises the steps of:
• the method according to the fifth aspect comprises the steps of:
• the method of the fifth aspect may be performed by directly producing, i.e. by synthesis, the plurality of RNA molecules.
• the method would comprise the steps of:
• the method according to the fifth aspect comprises the steps of:
• RNA molecules obtained or obtainable from the method defined in said aspect.
• the present invention also provides a DNA molecule comprising least one sequence that, upon transcription, generates an RNA molecule comprising a hammerhead type I ribozyme in its 5' end and a hammerhead type III ribozyme in its 3' end.
• the hammerhead type I ribozyme placed in the 5' end is the self-catalytic polyribonucleotide of the invention.
• the hammerhead type III ribozyme placed in the 3' end is the Elvd ribozyme, preferably comprising or consisting of SEQ ID NO: 32.
• This DNA molecule can also be used as a precursor in a method to generate circular RNAs, since the RNA molecule comprising a hammerhead type I ribozyme in its 5' end and a hammerhead type III ribozyme in its 3' end will be self-cleaved, and a RNA molecule with ligatable ends will be obtained.
• the sixth aspect of the invention also refers to a method to generate circular RNAs, the method comprising the steps of:
• the sixth aspect of the invention also refers to a method to generate circular RNAs, the method comprising the steps of:
• the sixth aspect of the invention also refers to a method to generate circular RNAs, the method comprising the steps of:
• the sixth aspect of the invention also refers to a method to generate circular RNAs, the method comprising the steps of:
• RNA molecule obtained or obtainable from the method defined in said aspect.
• the three precursor circRNA containing our three artificial 5'-HH self-cleaving ribozymes (encoded by SEQ ID NO 16-18; in lanes 5-7) designed showed a double self-cleavage resulting in a RNAfragment of approximately 300 nt.
• the construct containing previously described naturally occurring HH from Ricordea florida (SEQ ID NO 12, lane 1) is also double self-cleaved forming the 300 nt circRNA fragments, however, a less intense band is shown in the polyacrylamide-urea gel suggesting a lower self-cleavage efficiency.

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